Dissecting The Role Of GHSR1a In Regulating Fear Memory Generalization Of Stressed Mice

Memory is a process of encoding,consolidating,storing and retrieving acquired information.Fear memories occur when the individuals experience traumatic events.However,as time goes by,the specificity of fear memory gradually weakens,meanwhile the generalization of fear memory appears.Memory generalization means that,after leaning a conditioned stimulus for a period of time,the individuals start to show conditioned responses to other stimuli sharing similarity to the original conditioned one.Contextual fear is a common behavioral paradigm to study memory generalization.In recent years,more and more researches have focused on fear memory generalization.The hippocampus is the main brain region to store contextual fear memories.Specifically,the hippocampus encodes and replays detailed information of recent events,and then projects this information to the cortex.Some details lose during contextual fear memories are transferring from the hippocampus to the cortex,herein generalization occurs.Post-traumatic stress disorder(PTSD)is an emotional disorder that developed after an individual is exposed to traumatic events leading to persistent physical and mental dysfunction.Overgeneralization of fear memory is considered the most important characteristic of fear-related disorder or PTSD.Studies have demonstrated that stress exposure occurred before the traumatic event was also a risk factor for the development of PTSD.Stress events can trigger the release of stress hormones,neurotransmitters and neuropeptides.Some evidence have illustrated that stress and stress-related hormones enhance the generalization of fear memory.Previous studies showed that fear memory generalization could be regulated by factors such as threats intensity,stimulus similarity,and perception.In addition,fear memory generalization requires a complex balance of excitation and inhibition in different brain regions.However,the mechanism of fear memory overgeneralization is still unknown.Growth hormone secretagogue ghrelin is a 28-amino acids peptide mainly secreted by the stomach and perhaps by the hypothalamus.Ghrelin plays an important role in energy metabolism and eating control.Only acylated ghrelin by ghrelin-O-acyltransferase(GOAT)can activate its receptor growth hormone secretagogue receptor 1a(GHSR1a).GHSR1a distributes both inside and outside the brain,with enriched expression in the central nervous system including the hypothalamus,the hippocampus,the cortex,the amygdala,the substantia nigra and many other brain regions.Studies have shown that the ghrelin/GHSR1a system not only regulates food intakes and energy metabolism,but also plays complex role in regulating learning and memory,stress response,motivation and reward.Remarkably,the expression of ghrelin and GHSR1a were upregulated in multiple brain regions after stress,especially after chronic stress exposure.Some studies have thus suggested that ghrelin was not only a feeding-control hormone,but also a stress hormone.Previous studies in our lab also showed that GHSR1a played an important role in regulating hippocampus-dependent memory acquisition and anxiety-related behaviors.GHSR1a deletion promoted the formation of fear memory;meanwhile it reduced anxiety-related behaviors.The pattern-separating function and the adult neurogenesis in DG region of the hippocampus are thought to be involved in memory generalization process.GHSR1a is abundant in DG region,but the role of ghrelin/GHSR1a in memory generalization has not been reported yet.We aimed in this study to explore the role of GHSR1a in fear memory generalization.Our study may help better understand the complex functions of the ghrelin/GHSR1a system in CNS,and provide a new potential target for the prevention and the treatment of cognitive and affective dysfunction associated with PTSD.In this study,GHSR1a knockout mice,virus-mediated GHSR1a overexpressing mice and control WT mice were subjected to different patterns of stress exposure,including single prolonged stress(SPS),repeated restrict stress(RRS),and intraperitoneal injection of lipopolysaccharide(LPS).Effects of different stress exposure on the generalization of fear memory as well as the role of ghrelin/GHSR1a signaling in this process were evaluated with contextual fear conditioning paradigm.We found that,1.SPS exposure increased hippocampal Ghsr1a expression and promoted anxiety-like behaviors in mice.2.SPS exposure enhanced 24-h contextual fear memory in WT mice.Specifically,the stressed mice showed higher level of freezing in shock context than non-stressed control mice.In addition,both groups of mice showed higher freezing in the shock context than in the novel context.3.SPS exposure led to significant generalization of contextual fear memory at 7 daysafter conditioning in WT mice.The stressed mice showed similar freezing in shock context as in novel context.4.RRS exposure also increased Ghsr1a expression in the hippocampus,andpromoted anxiety-like behaviors in WT mice.However,RRS had no significant effect on either fear memory formation or fear memory generalization.Selective overexpression of GHSR1a in DGαCa MKII~+neurons in the hippocampus did not affect either fear memory formation or fear memory generalization in RRS stressed mice.5.LPS injection had no effect on hippocampal Ghsr1a,neither did it affect fearmemory.6.GHSR1a KO enhanced 1day fear memory of non-stressed mice.The non-stressedKO mice showed higher freezing in shock context than non-stressed WT mice.However,GHSR1a KO suppressed fear memory of SPS-stressed mice.The stressed KO mice showed less freezing in shock context than stressed WT mice.7.Different from SPS-exposed WT mice,SPS-exposed GHSR1a KO mice exhibitedhigher freezing in shock context than in novel context 7 days after conditioning,which indicated no generalization of fear memory in KO mice.These findings suggest that GHSR1a deficiency suppresses generalization of fear memory;in other words,SPS-induced upregulation of endogenous Ghsr1a in the hippocampus may promote fear memory generalization.8.Selective overexpression of GHSR1a in DG DLX5/6~+inhibitory neurons in thehippocampus did not affect the formation of fear memory in SPS-exposed mice,but inhibited fear memory generalization in those mice.Specifically,GHSR1a-overexpressing mice exhibited higher freezing in shock context than in novel context 7 days after conditioning,while control mice without GHSR1a overexpression showed similar freezing in two contexts.In summary,we demonstrate that SPS-stress upregulates Ghsr1a expression in the hippocampus and promotes fear memory generalization in stressed mice.Both GHSR1a deletion and GHSR1a overexpression in DG DLX5/6~+inhibitory neurons inhibited fear memory generalization in SPS-exposed mice.We conclude that SPS-induced GHSR1a upregulation in the hippocampus contributes to the generalization of fear memory,and this regulation is neuron-type specific.Altogether,our findings shed light on the role of GHSR1a on stress-induced fear memory generalization,and further studies are still required to elucidate the precise mechanism underlying this process.